The crosstalk between glomerular endothelial cells and podocytes controls their responses to metabolic stimuli in diabetic nephropathy

In diabetic nephropathy (DN), glomerular endothelial cells (GECs) and podocytes undergo pathological alterations, which are influenced by metabolic changes characteristic of diabetes, including hyperglycaemia (HG) and elevated methylglyoxal (MGO) levels. However, it remains insufficiently understood what effects these metabolic factors have on GEC and podocytes and to what extent the interactions between the two cell types can modulate these effects. To address these questions, we established a co-culture system in which GECs and podocytes were grown together in close proximity, and assessed transcriptional changes in each cell type after exposure to HG and MGO. We found that HG and MGO had distinct effects on gene expression and that the effect of each treatment was markedly different between GECs and podocytes. HG treatment led to upregulation of “immediate early response” genes, particularly those of the EGR family, as well as genes involved in inflammatory responses (in GECs) or DNA replication/cell cycle (in podocytes). Interestingly, both HG and MGO led to downregulation of genes related to extracellular matrix organisation in podocytes. Crucially, the transcriptional responses of GECs and podocytes were dependent on their interaction with each other, as many of the prominently regulated genes in co-culture of the two cell types were not significantly changed when monocultures of the cells were exposed to the same stimuli. Finally, the changes in the expression of selected genes were validated in BTBR ob/ob mice, an established model of DN. This work highlights the molecular alterations in GECs and podocytes in response to the key diabetic metabolic triggers HG and MGO, as well as the central role of GEC-podocyte crosstalk in governing these responses.


SUPPLEMENTARY FIGURES
Figure S1.HG and MGO do not significantly affect the number of GEC or podocytes.(A-D) GECs (A, C) and podocytes (B, D) were differentiated and treated with 25 mM glucose (HG, addition of 19.5 mM glucose to RPMI-1640 medium containing 5.5 mM glucose, A, B) or with 200 µM MGO (C, D) for 96 hr as described (see Fig. 1A).5.5 mM glucose + 19.5 mM mannitol (osmotic control) and water served as treatment controls, respectively.Cell viability / cell numbers were assessed using a CyQUANT Proliferation Assay and the fluorescence signal was normalised relative to the control condition (n=3-4).S3.Forward and reverse primer sequences used for qPCR.

Figure S2 .
Figure S2.MGO has limited effects on podocytes.(A) Volcano plot of gene expression changes in podocytes exposed to 96 hr of MGO compared to control cells.Genes with log2FC >1 and FDRcorrected p-values <0.05 were marked in red (strongly upregulated genes).(B) Heat maps of all up-and downregulated genes in GECs after 96 hr of MGO.

Figure S3 .
Figure S3.Limited overlap between the genes regulated by HG in podocytes and by MGO in GECs.Venn diagrams showing the overlaps between DEGs in podocytes exposed to HG and GECs exposed to MGO for 96 hr.Upregulated genes sets are represented by red hues, downregulated gene sets -by blue hues.

Figure S4 .
Figure S4.MGO treatment of GECs increases ID3 and decreases FN1 protein levels in co-cultures with podocytes, but not in monocultures.(A, B) GECs were co-cultured with podocytes as described (see Fig.1A) and exposed to 200 µM MGO for 96 hr; water served as treatment control.GECs were lysed and analysed by Western blot to determine the protein levels of ID3 (A) and FN1 (B).Actin and tubulin served as loading controls.The graphs under the Western blots present densitometric quantification of protein levels normalised to loading controls for the separate replicates (n=3).(C) GECs were cultivated in monoculture, exposed to 200 µM MGO for 96 hr and the protein levels of ID3 and FN1 were determined by Western blot.The graphs next to the Western blots present densitometric quantification of protein levels normalised to loading controls for the separate replicates (n=3).

Figure S5 .
Figure S5.Co-staining for ID3 and glomerular endothelial cells or podocytes in BTBR wt/wt and ob/ob animals.(A) Kidney sections of BTBR ob/ob and control (BTBR wt/wt) mice were co-stained using immunofluorescence for ID3 (green), CD31 as an endothelial cells marker (magenta) and DAPI to visualise nuclei (cyan).The ID3 and CD31 channels, as well as the ID3/CD31/DAPI channels are shown as overlays on the right, as indicated above the images.(B) Kidney sections of BTBR ob/ob and control (BTBR wt/wt) mice were co-stained using immunofluorescence for ID3 (green), nephrin as a podocyte marker (magenta) and DAPI to visualise nuclei (cyan).The ID3 and nephrin channels, as well as the ID3/nephrin/DAPI channels are shown as overlays on the right, as indicated above the images.Shown are single optical slices from confocal stacks.Scale bars, 20 µm.

Figure S6 .
Figure S6.Original images of the full membranes for the Western blots shown in Figure S4.Panels A, B and C present images of the full membranes for the Western blots shown in Figure S4A, B and C, respectively.Shown on the left are photographic images of the membranes overlaid with an exposure of the chemiluminescence signal.The exposure used for assembling the corresponding panels in Figure S4 are shown to the right with the same field of view, but containing the chemiluminescence signal only.The dotted rectangles over the Western blots indicate the cropped regions used in Figure S4.The numbers to the left indicate the molecular weight of the markers that can be seen on the membranes at the respective height.The names of the antigens are indicated to the right of the Western blots.